This renewal proposal has as its long-term objective the elucidation of molecular mechanisms of normal and abnormal expression of genes encoding proteins of the membrane skeleton, in particular the genes for spectrin and ankyrin which are important constituents of the membrane skeleton of erythrocytes as well as non- erythroid cells. Study of these genes is of scientific interest for a number of reasons: I) several human diseases are associated with mutations in these genes; II) membrane proteins analogous to those in red cells but having a somewhat different structure are expressed in non-erythroid cells, and the molecular sources of their multiple isoforms as well as the regulation of their temporal and spatial expression is unknown; and III) the structure/function relationships of interacting membrane skeleton proteins is still not completely understood and warrants further investigation. Specific aims designed to clarify these issues include:I) completion of the cloning and DNA sequence analysis of selected regions of the chromosomal spectrin, fodrin and ankyrin genes, with emphasis on promoter regions and regions thought to be involved in alternative splicing; II) characterization of cis-acting elements and trans-acting factors involved in tissue-specific expression of these genes; III) characterization of mutations causing abnormal spectrin, ankyrin and band3 expression, with special emphasis on identifying mutations responsible for hereditary spherocytosis; and IV) analysis of structure-function relationships of selected recombinant spectrin and fodrin domains, e.g. the SH3 domain of alpha-spectrin. Major experimental methods to be employed include: cloning and structural analysis of the cDNAs and genomic DNA segments of the relevant genes by use of recombinant DNA technology; study of the DNA from individuals with different hereditary disorders of these genes by use of the polymerase chain reaction (PCR) technique; studies of cis-acting sequences by gene manipulation followed by gene transfer/expression studies in tissue culture cells; studies of trans- acting factors by gelretardation assays, footprinting and methylation interference techniques; studies of structure/function relationshipsby production of recombinant proteins in various expression systems and analysis of their function in biochemical and/or biological test systems.